Medicaments

ABSTRACT

A drug conjugate comprising a targeting agent and an anti-cancer agent, wherein said targeting agent comprises an erythropoietin receptor ligand, is described. The drug conjugate can be used in methods of treating cancer. Also described are methods of treating cancer using the conjugate, methods of diagnosis, methods of imaging and pharmaceutical compositions.

FIELD OF THE INVENTION

This application relates to a medicament and its use in methods oftreatment, diagnosis and imaging. In particular, it relates to thetreatment of cancer with a cytotoxic agent linked to a targeting agent.

BACKGROUND TO THE INVENTION

This invention relates to anti-cancer medicaments that are specificallytargeted to cancer cells by being linked to Erythropoietin (EPO)receptor ligands.

It is widely known in the field of oncology that the main disadvantageof chemotherapy is its inherent lack of specificity. Althoughanti-cancer drugs like Taxol™ are very successful in arresting cellulardivision in tumour cells, they also have the same effect on normalcells, giving rise to significant tissue and organ lesions, some ofwhich are irreversible. Hence, it is essential to overcome thisunspecific nature of chemotherapy so that the patient can tolerate itmore and increase the efficiency of the drugs used.

EPO is a 30.4 kD Glycoprotein Hormone that is the main growth factorresponsible for the regulation of red blood cell production in mammals(Erythropoiesis).

The primary site for the production of EPO in adult organisms is thekidney, although lower levels of EPO are synthesised by the liver andbrain.

Synthesis of EPO itself in the body is regulated by oxygen tension inthe body tissues, and is controlled by both positive and negativefeedback signals. Low oxygen tension induces EPO production which inturn causes an increase in red blood cell production in the bone marrow.The enhanced oxygen supply to body tissues reduces EPO synthesis.

Recombinant human EPO is now readily available and is regularly used inthe treatment of anaemia resulting from renal failure, harsh drugtreatment like chemotherapy, and HIV-infection. The protein sequence forEPO, 193 amino acids in length, can be found in GenBank under AccessionNo. 1104303A (Jacobs et al. (1995) Nature 313,806-810), which isincorporated herein by reference in its entirety.

The protein sequence for the erythropoietin receptor, 508 amino acids inlength, can be found in GenBank, under Accession No. AAA52403 (Jones etal. (1990) Blood 76,3135), which is also incorporated herein byreference in its entirety.

EPO circulates in the plasma at low concentrations (in pico-molar range)and binds to EPO-receptors (EPO-R) situated on the cell surfacemembrane. For many cells the receptor number is low, ranging fromseveral hundred to several thousand of EPO-R per cell. Other cells canhave high numbers of EPO-R in the order of 30,000 or greater.

The EPO Receptor is a 55 kD, (508 amino acid residue) transmembraneprotein comprised of a 24 amino acid signal peptide, a 226 amino acidexternal segment, a 22 amino acid transmembrane segment, and a 236 aminoacid cytoplasmic domain. This receptor is activated via a single EPOmolecule bridging EPO receptor pairs. Binding of EPO to these EPOreceptors causes a cascade of events including phosphorylation of theprotein tyrosine kinase, Jak2. This in itself induces phosphorylation ofthe EPO receptors at 8 tyrosine residues. A vast array of other proteinkinase signalling events are subsequently involved which ultimatelycause the cell to express proteins involved in Erythropoiesis.

Recently Acs et al, Cancer Research 61, 3561-3565, 2001, reported thathuman breast cancer cells express the Erythropoietin (EPO) receptor.This result is confirmed herewith, but we surprisingly discloseadditionally that other human tumours express the EPO receptor, whichhas led to the present invention.

SUMMARY OF THE INVENTION

As described herein, the present inventors have surprisingly shown thaterythropoietin receptor expression is upregulated in cancer cells otherthan breast cancer cells. In particular, the inventors have shown thaterythropoietin receptors are expressed in cells of lung cancer and nonsmall cell lung cancers.

The demonstration that erythropoietin receptors are expressed on tumourcells at a supranormal level enables new methods of detecting and/orkilling cancer cells in vitro or in vivo. In particular, it enables thetargetting of cancer cells in a tissue.

According to a first aspect of the present invention, there is provideda drug conjugate comprising a targeting agent and an anti-cancer agent,wherein said targeting agent comprises an erythropoietin receptorligand.

According to a second aspect of the invention, there is provided amethod of killing cancer cells comprising contacting said cancer cellswith a drug conjugate according to the first aspect of the invention.

The method of the second aspect of the invention can be used to killcancer cells in vitro or in vivo. Thus, a third aspect of the inventionprovides a method of treating cancer, said method comprisingadministration of a therapeutically effective amount of a drug conjugateaccording to the first aspect of the invention to a mammal in needthereof.

In a fourth aspect, there is provided the use of a drug conjugateaccording to the first aspect of the invention in the preparation of amedicament for treating cancer.

According to a fifth aspect, there is provided a pharmaceuticalcomposition for the treatment of cancer, wherein the compositioncomprises a drug conjugate according to the first aspect of theinvention and a pharmaceutically acceptable excipient, diluent orcarrier.

The presence of erythropoietin receptors on a tissue may be useful forthe diagnosis of cancer or monitoring of progression of cancer. This mayinvolve determining the number of EPO-receptors (and/or one or moreisoforms and/or mutants of the erythropoietin receptor) on each cell ora population of cells.

Thus, according to a sixth aspect of the present invention, there isprovided a method of diagnosis of the presence of cancer cells in abiological sample, said method including the step of contacting a ligandwhich binds to erythropoietin receptors with said sample and detectingbinding of said ligand to cells of the sample.

In those tissues where erythropoietin receptors are not present onnormal, non-cancerous cells, the detection of binding to erythropoietinreceptors is indicative of the presence of cancer cells. In those tissuein which erythropoietin receptors are normally present, an increase inthe level of expression is indicative of cancer.

The method of the sixth aspect of the invention may be performed invitro or in vivo. In in vivo methods, the invention may be used to imagetumours e.g. to detect the presence of or monitor the progression ofcancer.

Thus, in a seventh aspect of the invention there is provided a method ofimaging a cancer in a patient, said method comprising administering tothe patient an erythropoietin receptor ligand coupled to an imagingagent, allowing said erythropoietin receptor ligand to bind toerythropoietin receptors and detecting the imaging agent.

The imaging agent may be any suitable agent. For example, the agent maybe a paramagnetic ion or a radioisotope. For example, where the patientis imaged using positron emission tomography, the imaging agent may be apositron emitter such as fluorine 18.

In this aspect of the invention, the ligand is labelled with anysuitable imaging agent. Suitable imaging agents, e.g. for use withX-ray, PET etc are known in the art.

Indeed, in a further aspect of the present invention, there is providedan erythropoietin receptor ligand labelled by an imaging agent.

According to a further aspect of the current invention there is provideda medicament capable of avoiding efflux of the drug from the cancer cellby the Multi Drug Resistance (MDR) membrane glyco-protein, saidmedicament comprising a drug conjugate according to the first aspect ofthe invention. The problem of acquired resistance by tumour cells todrugs used in chemotherapy is a major problem today.

The drug conjugate, composition, medicament, uses and methods of theinvention may be used for the treatment or diagnosis of any cancer. Forexample, the invention may be used in cancers of breast, cervix, uterus,ovary, prostate, brain, stomach or lung. In particularly preferredembodiments of the first aspect of the invention, the sample is fromlung tissue. In a most preferred aspect of the present invention, thecancer is lung cancer and/or non-small cell lung carcinoma.

DETAILED DESCRIPTION

Drug Conjugates

As described above, the present invention provides a drug conjugatecomprising a targeting agent and an anti-cancer agent, wherein saidtargeting agent comprises an erythropoietin receptor ligand. Anysuitable drug conjugate may be used in the present invention. Drugconjugates and their effects may be tested using conventional methods.Known methods of identifying, verifying and testing ideal anticanceragent-ligand complexes for the EPO-receptors include Elisa and SurfacePlasmon Resonance, e.g. Bia-Core™, dimerisation of EPO receptors can beused to give an indication of the functionality of the drug-ligandcomplex.

For example, Daunorubicin, may be chemically linked to an EPO receptorligand, the EPO mimetic peptide EMP-1 for example.

Medicaments/conjugates may be screened using known methods, e.g. Elisaand Surface Plasmon Resonance, by analysing their binding to EPOreceptors and inducing dimerisation of EPO receptor pairs. Preferablythe drug-ligand complex is non-immunogenic.

Binding of the drug-ligand can cause sequestration of the EPO-receptorwith its bound ligand and the linked drug into the cell cytoplasm,whereafter the ligand and its linked drug may be released from thereceptor.

DETAILED DESCRIPTION

Ligands

In the context of the present invention, a “ligand” is a molecule whichhas binding specificity for another molecule, in particular theerythropoietin receptor. The ligand may be a member of a pair ofspecific binding members. The members of a binding pair may be naturallyderived or wholly or partially synthetically produced. One member of thepair of molecules may have an area on its surface, which may be aprotrusion or a cavity, which specifically binds to and is thereforecomplementary to a particular spatial and polar organisation of theother member of the pair of molecules. Thus, the members of the pairhave the property of binding specifically to each other. Examples oftypes of binding pairs are antigen-antibody, biotin-avidin,hormone-hormone receptor, receptor-ligand, enzyme-substrate.

The present invention is concerned with receptor-ligand type reactions,although a binding member of the invention and for use in the inventionmay be any moiety, for example an antibody, which can bind to anerythropoietin receptor.

In preferred embodiments of the invention, the ligand is erythropoietin(EPO), an EPO analogue or fragment thereof or a functional mimetic ofEPO.

An analogue of EPO or a fragment thereof means any polypeptide orantibody modified by varying the amino acid sequence of the EPO protein,e.g. by manipulation of the nucleic acid encoding the protein or byaltering the protein itself. Such derivatives of the natural amino acidsequence may involve insertion, addition, deletion and/or substitutionof one or more amino acids, preferably while providing a peptide havingEPO activity, for example, erythropoietin receptor binding activity.Preferably such analogues involve the insertion, addition, deletionand/or substitution of 25 or fewer amino acids, more preferably of 15 orfewer, even more preferably of 10 or fewer, more preferably still of 4or fewer and most preferably of 1 or 2 amino acids only.

Functional mimetics of EPO are substances which are not necessarilypeptides and need not necessarily comprise the active portion of theamino acid sequence of EPO but which nevertheless retain biologicalactivity of EPO.

A typical mimetic which may be used in the present invention is EMP-1,as described by Connolly et al, Bioorganic & Medicinal Chemistry Letters10, 2000, 1995-1999.

Antibodies

The ligand may be an antibody. An “antibody” is an immunoglobulin,whether natural or partly or wholly synthetically produced. The termalso covers any polypeptide, protein or peptide having a binding domainwhich is, or is homologous to, an antibody binding domain. These can bederived from natural sources, or they may be partly or whollysynthetically produced. Examples of antibodies are the immunoglobulinisotypes and their isotypic subclasses and fragments which comprise anantigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies.

The ligand of and for use in the invention may be an antibody such as amonoclonal or polyclonal antibody, or a fragment thereof. The constantregion of the antibody may be of any class including, but not limitedto, human classes IgG, IgA, IgM, IgD and IgE. The antibody may belong toany sub class e.g. IgG1, IgG2, IgG3 and IgG4.

As antibodies can be modified in a number of ways, the term “antibody”should be construed as covering any binding member or substance having abinding domain with the required specificity. Thus, this term coversantibody fragments, derivatives, functional equivalents and homologuesof antibodies, including any polypeptide comprising an immunoglobulinbinding domain, whether natural or wholly or partially synthetic.Chimeric molecules comprising an immunoglobulin binding domain, orequivalent, fused to another polypeptide are therefore included. Cloningand expression of chimeric antibodies are described in EP-A-0120694 andEP-A-0125023.

Fragments of a whole antibody can perform the function of bindingantigens. Examples of such binding fragments are (i) the Fab fragmentconsisting of VL, VH, CL and CH1 domains; (ii) the Fd fragmentconsisting of the VH and CH1 domains; (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward, E. S. et al., Nature 341:544-546 (1989)) which consists of a VHdomain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalentfragment comprising two linked Fab fragments (vii) single chain Fvmolecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., Science 242:423-426 (1988); Huston etal., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

A fragment of an antibody or of a polypeptide for use in the presentinvention generally means a stretch of amino acid residues of at least 5to 7 contiguous amino acids, often at least about 7 to 9 contiguousamino acids, typically at least about 9 to 13 contiguous amino acids,more preferably at least about 20 to 30 or more contiguous amino acidsand most preferably at least about 30 to 40 or more consecutive aminoacids.

The term “antibody” includes antibodies which have been “humanised”.Methods for making humanised antibodies are known in the art. Methodsare described, for example, in Winter, U.S. Pat. No. 5,225,539. Ahumanised antibody may be a modified antibody having the hypervariableregion of a monoclonal antibody and the constant region of a humanantibody. Thus the binding member may comprise a human constant region.

The variable region other than the hypervariable region may also bederived from the variable region of a human antibody and/or may also bederived from a monoclonal antibody. In such case, the entire variableregion may be derived from murine monoclonal antibody and the antibodyis said to be chimerised. Methods for making chimerised antibodies areknown in the art. Such methods include, for example, those described inU.S. patents by Boss (Celltech) and by Cabilly (Genentech). See U.S.Pat. Nos. 4,816,397 and 4,816,567, respectively.

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementary determining regions(CDRs), of an antibody to the constant regions, or constant regions plusframework regions, of a different immunoglobulin. See, for instance,EP-A-184187, GB 2188638A or EP-A-239400. A hybridoma or other cellproducing an antibody may be subject to genetic mutation or otherchanges, which may or may not alter the binding specificity ofantibodies produced.

Anti-Cancer Agents

Any suitable chemotherapeutic agent or agents may be used as theanti-cancer agent in the present invention.

The agent may be any kind of drug or peptide or piece of DNA or RNA thatinhibits cellular division, or causes apoptosis.

For example, the agent for use in the invention may include but is notlimited to: Anthracycline Antibiotic, Daunorubicin, Doxorubicin, Taxol™,5-Fluorouracil (5 FU), Leucovorin, Irinotecan, Idarubicin, Mitomycin C,Oxaliplatin, Raltitrexed, Tamoxifen and Cisplatin, Actinomycin D,Mitoxantrone or Blenoxane or Mithramycin.

In one preferred embodiment, the agent is a DNA chelator. Morepreferably the agent is an

Anthracycline Antibiotic. Even more preferably the drug is Doxorubicinor Idarubicin. Most preferably the drug is Daunorubicin.

In another preferred embodiment, the agent is Actinomycin D Mitoxantroneor Blenoxane or Mithramycin.

In another preferred embodiment the agent is a cytoskeletal bindingdrug. Even more preferably the drug is paclitaxel (Taxol™).

The agent can also be a member of the Bio-Reductive drugs that areactivated under hypoxic cellular conditions.

Suitable BioReductive drugs include SR4233 or AQ4N.

The ligand may be linked to one or more anti-cancer agent molecules,which may be the same or different.

Preferably the ligand has more than one agent molecule linked to it.

Linkers

The anti-cancer agent may be linked to the ligand by any suitable means.For example, the anti-cancer agent-ligand link may be an amine bond. Inone embodiment, the drug-ligand link comprises two or more amino acids.

More preferably the drug-ligand link is via bi-functional chemical crosslinkers, like Pierce™ DSP, DVP and the like.

Preferably, the drug-ligand amino acid link is cleavable, for example anester band, cleavable by intracellular esterases.

Preferably the link is cleavable under low oxygen tensions, i.e.sensitive to the cytoplasmic REDOX state, in order to increase thetoxicity of the drug in the cell.

For the purpose of this invention, “linker” is defined as a chemicalcompound which can form a covalent bond with an anti-cancer agent andanother covalent bond with the ligand on the other hand. Preferably, alinker used in practising the present invention should have a suitablelength and should not have a significant effect on the anticanceragent's therapeutic property or the erythropoietin receptor ligand'sspecific affinity to the erythropoietin receptors on the targeted tumorcells.

Of course, the choice of a linker in a specific practice depends on whattype of anti-cancer drug it is to be conjugated to. For example, if theanti-cancer drug has a —OH group or a —NH₂ group for connecting alinker, the linker should preferably have a —COOH group so that an esterbond can be formed between the —OH and —COOH groups or a peptide bondcan be formed between the —NH₂ and —COOH; if the anti-cancer drug has a—COOH group for connecting a linker, then the linker should preferablyhave a free —NH₂ group (in addition to the —NH₂ group which forms apeptide bond with the erythropoietin receptor ligand) so that a peptidebond can be formed between the —COOH and —NH₂ groups.

If the anti-cancer drug has a maleimide group, the linker shouldpreferably have a free —SH group so that a covalent S-maleimide bond canbe formed (and, vice versa, if the anti-cancer agent has an —SH group,the linker should have a maleimide group).

The conjugation between a maleimide group and a sulfhydryl group (—SH)has an additional advantage because the overall synthesis yield isincreased as the conjugation can be conducted after the peptide cleavageand deprotection. This can prevent the anti-cancer drug from TFAtreatment in the peptide cleavage and deprotection step.

In a preferred embodiment of the invention, maleimide is used as alinker group. In one preferred embodiment, the maleimide group isattached to the 3′ amino position of the anti-cancer agent daunorubicin,e.g. via a benzamide bond. In another preferred embodiment, themaleimide group is attached to the 13 keto group of the anti-canceragent daunorubicin.

In further embodiments, the formation of stable, covalently linkedconjugates with fully retained biological activities of an anticancerdrug may be achieved by using a di-carboxylic acid linker, such asglutaric acid. One carboxyl group of the linker group forms an esterbond with the anticancer agent e.g the 2′-OH group of paclitaxel or the—OH groups of other anticancer drugs and the other carboxyl group of thelinker group forms a carboxamide bond with a well chosen free aminogroup of the peptide carrier, such as an erythropoietin analogue.

Because some anti-cancer drugs, such as paclitaxel, have poor watersolubility, a conjugated drug-linker-erythropoietin receptor ligandcomplex can have a free —NH₂ group for further connection to a componentwhich can improve the drug's water solubility. For instance, the —NH₂can be connected to a PEG, sugar or biotin group Moreover theerythropoietin receptor may be linked to the anticancer agent indirectlye.g. via liposomes, where the erythropoietin receptor ligand iscovalently connected to a compound which then forms liposomes, where oneor more anti-cancer agents can be disposed. The anticancer agent in theliposomes will be released once the conjugated erythropoietin receptorligand brings the liposomes to the targeted tumor cells.

Treatment

“Treatment” includes any regime that can benefit a human or non-humananimal. The treatment may be in respect of an existing condition or maybe prophylactic (preventative treatment). Treatment may includecurative, alleviation or prophylactic effects.

“Treatment of cancer” includes treatment of conditions caused bycancerous growth and includes the treatment of neoplastic growths ortumours. Examples of tumours that can be treated using the inventionare, for instance, sarcomas, including osteogenic and soft tissuesarcomas, carcinomas, e.g., brain, breast-, lung-, bladder-, thyroid-,prostate-, colon-, rectum-, pancreas-, stomach-, liver-, uterine-,cervical and ovarian carcinoma, lymphomas, including Hodgkin andnon-Hodgkin lymphomas, neuroblastoma, melanoma, myeloma, Wilms tumor,and leukemias, including acute lymphoblastic leukaemia and acutemyeloblastic leukaemia, gliomas and retinoblastomas.

In preferred embodiments, the present invention is used and is usefulfor the treatment of one or more of breast cancer, cancer of the kidney,brain cancers, lung cancer and non small cell lung carcinoma. Inparticularly preferred embodiments, the present invention is used and isuseful for the treatment of lung cancer and/or non small cell lungcarcinoma.

The conjugates, compositions and methods of the invention may beparticularly useful in the treatment of existing cancer and in theprevention of the recurrence of cancer after initial treatment orsurgery.

Administration

Conjugates and compositions of and for use in the present invention maybe administered to a patient in need of treatment via any suitableroute. The precise dose will depend upon a number of factors, includingthe precise nature of the conjugate and and/or anticancer agent and/orthe nature and site of the cancer.

Some suitable routes of administration include (but are not limited to)oral, rectal, nasal, topical (including buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous,intradermal, intrathecal and epidural) administration. Intravenousadministration is preferred.

It is envisaged that injections (intravenous) will be the primary routefor therapeutic administration of conjugates and compositions althoughdelivery through a catheter or other surgical tubing is also envisaged.Liquid formulations may be utilised after reconstitution from powderformulations.

In one embodiment, where, for example, the conjugates or compositionsare used for the treatment of bladder cancer, it is envisioned that theconjugate or composition is administered using a bladder wash technique.

In a further preferred embodiment, in particular for the treatment ofcancers of the lung, the conjugate is formulated for delivery viaaerosol, so that the drug-ligand complex can access the EPO receptorsfacing into the lumen of the bronchus or lungs. This prevents the needfor introducing excessive amounts of EPO or ligand into the bloodstream, which would down-regulate EPO receptor synthesis byerythropoietic cells.

Where the cancer is of the GI tract, the drug-ligand conjugate ispreferably ingested to access the EPO receptors facing the lumen of thedigestive tract.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pH, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

In a preferred embodiment, they are administered as a pharmaceuticalcomposition, which will generally comprise a suitable pharmaceuticalexcipient, diluent or carrier selected dependent on the intended routeof administration.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded.

The conjugate or composition may also be administered via microspheres,liposomes, other microparticulate delivery systems or sustained releaseformulations placed in certain tissues including blood. Suitableexamples of sustained release carriers include semipermeable polymermatrices in the form of shared articles, e.g. suppositories ormicrocapsules. Implantable or microcapsular sustained release matricesinclude polylactides (U.S. Pat. No. 3,773,919; EP-A-0058481) copolymersof L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al,Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) orethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15:167-277, 1981, and Langer, Chem. Tech. 12:98-105, 1982). Liposomes areprepared by well-known methods: DE 3,218, 121A; Epstein et al, PNAS USA,82: 3688-3692, 1985; Hwang et al, PNAS USA, 77: 4030-4034, 1980;EP-A-0052522; E-A-0036676; EP-A-0088046; EP-A-0143949; EP-A-0142541;JP-A-83-11808; U.S. Pat. Nos. 4,485,045 and 4,544,545. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. % cholesterol, theselected proportion being adjusted for the optimal rate of theconstituent leakage.

Examples of the techniques and protocols mentioned above and othertechniques and protocols which may be used in accordance with theinvention can be found in Remington's Pharmaceutical Sciences, 16thedition, Oslo, A. (ed), 1980.

The conjugate or composition may be administered in a localised mannerto a tumour site or other desired site.

Pharmaceutical Compositions

As described above, the present invention extends to a pharmaceuticalcomposition for the treatment of cancer, the composition comprisingcomposition comprises a drug conjugate according to the first aspect ofthe invention and a pharmaceutically acceptable excipient, diluent orcarrier. Pharmaceutical compositions according to the present invention,and for use in accordance with the present invention may comprise, inaddition to active ingredients, a pharmaceutically acceptable excipient,carrier, buffer stabiliser or other materials well known to thoseskilled in the art.

Such materials should be non-toxic and should not interfere with theefficacy of the active ingredient. The precise nature of the carrier orother material will depend on the route of administration, which may beoral, or by injection, e.g. intravenous.

The formulation may be a liquid, for example, a physiologic saltsolution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilisedpowder.

Dose

The conjugates or compositions are preferably administered to anindividual in a “therapeutically effective amount”, this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated. Prescription oftreatment, e.g. decisions on dosage etc, is ultimately within theresponsibility and at the discretion of general practitioners and othermedical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.

The optimal dose can be determined by physicians based on a number ofparameters including, for example, age, sex, weight, severity of thecondition being treated, the active ingredient being administered andthe route of administration.

It is anticipated that in embodiments of the invention the conjugate orcomposition could be given in combination with other forms ofchemotherapy or indeed radiotherapy. The conjugates and/or compositionsof the invention may be administered simultaneously, separately orsequentially with the other form of chemotherapy or radiotherapy.

The invention will now be described further in the followingnon-limiting examples. Reference is made to the accompanying drawings inwhich:

FIG. 1 a is a micrograph of a Breast Cancer sample section stainingpositive (HRPO) for the EPO Receptor at the periphery of the cell

FIG. 1 b is a negative control, using the secondary antibody only(Rabbit anti-mouse) antibody.

FIG. 2 a is a micrograph of a section from a human lung cancer biopsy,staining positive for the EPO receptor in the epithelial cilia; and

FIG. 2 b is a micrograph of a serial section negative control from thesame biopsy sample as in FIG. 2 a; and

FIG. 3 a is a micrograph of a Non Small Cell Lung Cancer also showingspecific staining for the EPO receptor; and

FIG. 3 b is a serial section negative control of the sample of FIG. 3 a;and

FIG. 4 is a diagrammatic representation of the EPO-mimetic EMP-1; and

FIG. 5 is a diagramatic representation of the anti-cancer drugdaunorubicin.

EXAMPLE 1 Human tumour Cells Demonstrate Enhanced Expression of EPOReceptors

The following data demonstrate that human tumour cells express EPOreceptors and that this can be used as a novel way of selectivelytargeting drugs to cancer cells.

With reference to FIG. 1 a and FIG. 1 b, the tumour cells of FIG. 1 aare immunohistochemically positive for the EPO receptor and where thereis normal surrounding tissue, the cells are completely free of stain.FIG. 1 b is a negative control (i.e. without the primary rabbitanti-human EPO receptor antibody).

FIGS. 2 a, 2 b, 3 a and 3 b now demonstrate that EPO receptor expressionis not limited to breast cancer cells but is also found in BronchialEpithelium of a lung cancer, see FIG. 2 a, where EPO receptor expressionis found polarised to the upper compartment of the epithelium, i.e.cilia. Some nuclear crowding is apparent, but otherwise themorphological pattern is normal. FIG. 2 b is clearly negative for theEPO receptor.

Additionally, FIG. 3 a shows EPO receptor expression in a section form apatient with Non Small Cell Lung Carcinoma. Loss of cell polarity isevident in the malignant epithelial cells. Some of the tumour cells arecytoplasmically positive for the EPO receptor (EPO-R). Areas of necrosisalso show definite EPO-R positivity which may indicate macrophageinvolvement. FIG. 3 b is also clearly negative for the EPO receptor.

The lack of expression of EPO receptors by the surrounding normal tissueclearly demonstrates that drugs may be specifically targetted to cancercells by linking them to erythropoietin receptor ligands such as EPO orEPO fragments or mimetic peptides.

EXAMPLE 2 Production of Conjugates

a) 3′Amino Maleimide Daunorubicin Derivative-EPO Conjugate

A daunorubicin-EPO conjugate is prepared as follows. A maleimidederivative of daunorubicin is prepared by reacting daunorubicin.HCl with3-maleimidobenzoic acid chloride and two equivalents of triethylamine inTHF for 15 h at room temperature in the dark. Isolation of the compoundis performed by chromatography on silica gel (THF/hexane 3/1) andsubsequent chromatography on a LiChroPrepDiol column(Merck) (THF/hexane3/1) to obtain a pure sample of the daunorubicin maleimide derivative inwhich the maleimide group is at the 3′amino position of daunorubicin.

The daunorubicin derivative is then reacted with a mutant erythropoietincomprising an additional cysteine group for binding to the double bondof the maleimide group of the daunorubicin derivative to form the drugconjugate.

The drug-ligand complex is then tested for activity using cultured nonsmall cell lung carcinoma cells. The ability of the cells to endocytosethe conjugates are determined.

Binding of the conjugates to the EPO receptor on the surface of thecancer cell, followed by sequestration of the conjugate into the cellcytoplasm, and its release from the EPO receptor therein, enables theDaunorubicin drug to retain its activity and interchelate the DNA, formsDNA breaks and catalyse the formation of free radicals.

Other methods of verifying activity of the conjugate for theEPO-receptors include Elisa and Surface Plasmon Resonance, e.g.Bia-Core™, dimerisation of EPO receptors will give an indication of thefunctionality of the drug-ligand complex.

Incorporation of the medicaments into the cancer cells is carried outusing standard in vitro cell culture and radiolabelling techniques.Resistance to efflux via the MDR protein in cancer cell lines thatexpress this protein is also analysed using the above mentioned methods.

b) 13 Keto Maleimide Daunorubicin Derivative-EPO Conjugate

A 13-keto maleimide daunorubicin derivative-EPO conjugate is prepared asfollows. A maleimide derivative of daunorubicin is prepared by reactingdaunorubicin.HCl with a five-fold excess of 3-maleimidobenzoic acidhydrazide (trifluoroacetate salt) in anhydrous methanol, addition ofcatalytic amounts of CF₃COOH and stirring for 96 h at room temperaturein the dark. The products are precipitated by repeated addition ofacetonitrile. Analytical samples are obtained by crystallisation frommethanol/acetonitrile to obtain a pure sample of the daunorubicinmaleimide derivative in which the maleimide group is at the 13 ketoposition of daunorubicin.

The conjugate produced is tested as described for the 3′amino derivativeconjugate as described in part (a).

All documents referred to in this specification are herein incorporatedby reference. Various modifications and variations to the describedembodiments of the inventions will be apparent to those skilled in theart without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificpreferred embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes of carrying out theinvention which are obvious to those skilled in the art are intended tobe covered by the present invention.

1. A drug conjugate comprising a targeting agent and an anti-canceragent, wherein said targeting agent comprises an erythropoietin receptorligand.
 2. The drug conjugate according to claim 1, wherein saidanti-cancer agent is daunorubicin or a derivative thereof.
 3. The drugconjugate according to claim 1, wherein said anti-cancer agent is amaleimide derivative of daunorubicin.
 4. The drug conjugate according toclaim 1, wherein said erythropoietin receptor ligand is erythropoietin(EPO), an EPO analogue or fragment thereof or a functional mimetic ofEPO.
 5. A method of killing cancer cells, comprising contacting saidcancer cells with a drug conjugate according to claim
 1. 6. A method oftreating cancer, comprising administration of a therapeuticallyeffective amount of a drug conjugate according to of claim 1 to a mammalin need thereof.
 7. (canceled)
 8. A pharmaceutical composition for thetreatment of cancer, wherein the composition comprises a drug conjugateaccording to claim 1 and a pharmaceutically acceptable excipient,diluent or carrier.
 9. A method of diagnosis of the presence of cancercells in a biological sample, comprising contacting a ligand which bindsto erythropoietin receptors with said sample and detecting binding ofsaid ligand to cells of the sample.
 10. A method of imaging a cancer ina patient, comprising; administering to the patient an erythropoietinreceptor ligand coupled to an imaging agent; allowing saiderythropoietin receptor ligand to bind to erythropoietin receptors; anddetecting the imaging agent.
 11. The method of claim 10, wherein saidimaging agent is a paramagnetic ion or a radioisotope.
 12. The method ofclaim 10, wherein said cancer is imaged by positron emission tomography(PET).
 13. A medicament capable of avoiding efflux of the drug from thecancer cell by the Multi Drug Resistance (MDR) membrane glyco-protein,comprising a drug conjugate according to claim
 1. 14. The conjugateaccording to claim 1 wherein said cancer is lung cancer or non-smallcell lung carcinoma.
 15. The method according to claim 9, wherein saidcancer is lung cancer or non-small cell lung carcinoma.
 16. (canceled)17. The pharmaceutical composition according to claim 8, wherein saidcancer is lung cancer or non-small cell lung carcinoma.
 18. Themedicament according to claim 13, wherein said cancer is lung cancer ornon-small cell lung carcinoma.